Trypsin Digestion to Assess Triple Helix Stability in Collagens as Measure of Collagen Folding

Collagens play a critical role in biology and pathology, and mutations in collagen genes cause a variety of often debilitating severe genetic disorders. The identification of genetic variants can provide a molecular diagnosis, improve the ability to provide a prognosis, affect disease management, and enable investigation of causal mechanisms. However, for many identified missense variants in genetic analysis, it remains unclear if they are pathogenic or benign and whether they affect the collagen protein. Moreover, degradation of fibrillar collagen is also a feature of many common diseases. The triple helical nature of fibrillar collagen makes it relatively resistant to proteinases such as trypsin and pepsin but insults to the triple helix render it more susceptible. Trypsin digestion can thus serve as an insightful experimental approach for probing the structural integrity of fibrillar collagens, assessing the effects of genetic mutations, and studying functional properties of collagen under physiological and pathological conditions. Here, we describe a procedure in which secreted collagen has been isolated from fibroblast cells and subjected to trypsin digestion under various temperatures and conditions. Subsequent western blotting is then employed to visualize digestion patterns whereby a more rapid digestion indicates increased sensitivity to trypsin digestion as a measure of the protein folding quality of the triple helix. This simple biochemical method enables functional assessment of the impact of genetic variants and other insults that affect the triple helix and thus the function of collagen.